An “Off–On” Electrochemiluminescent Biosensor Based on DNAzyme-Assisted Target Recycling and Rolling Circle Amplifications for Ultrasensitive Detection of microRNA

Abstract

In this study, an off-on switching of a dual amplified electrochemiluminescence (ECL) biosensor based on Pb(2+)-induced DNAzyme-assisted target recycling and rolling circle amplification (RCA) was constructed for microRNA (miRNA) detection. First, the primer probe with assistant probe and miRNA formed Y junction which was cleaved with the addition of Pb(2+) to release miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences (S2). Afterward, bare glassy carbon electrode (GCE) was immersed into HAuCl4 solution to electrodeposit a Au nanoparticle layer (depAu), followed by the assembly of a hairpin probe (HP). Then, dopamine (DA)-modified DNA sequence (S1) was employed to hybridize with HP, which switching off the sensing system. This is the first work that employs DA to quench luminol ECL signal, possessing the biosensor ultralow background signal. Afterward, S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA. With rational design, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which could exhibit strongly catalytic toward H2O2, thus amplified the ECL signal and switched the ON state of the sensing system. The liner range for miRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM. Moreover, with the high sensitivity and specificity induced by the dual signal amplification, the proposed miRNA biosensor holds great potential for analysis of other interesting tumor markers.

A Review of Electrogenerated Chemiluminescent Biosensors for Assays in Biological Matrices

“…MicroRNA miRNA are a class of short, single-stranded noncoding RNA sequences, usually 19-23 nucleotides [53,54]. Despite their small sequence, miRNA are thought to play a significant role in regulation of fundamental cellular processes such as transcriptional and translational regulation of gene expression [53][54][55].…”

“…Despite their small sequence, miRNA are thought to play a significant role in regulation of fundamental cellular processes such as transcriptional and translational regulation of gene expression [53][54][55]. MiRNA-155, in particular, has been established as a biomarker for certain types of cancer.…”

“…Luminol-based ECL biosensors were used to detect the expression of miRNA-155 in cell lysates from various cell lines. These included human renal cubularepithelial cell line (HK-2), normal hepatocyte cell line (L02), human umbilical vein endothelial cell line and cervical cancer cell line (HeLa) (Figure 4) [53]. The technique first used dopamine (DA) to quench the luminol ECL signal.…”

“…It plays an important role in the initiation and progression of diabetic nephropathy (DN), and determining PDGF-BB levels can be useful in an early diagnosis of DN. PDGF also plays an [53] microRNA-155 HCR coupled with hemin intercalation as luminol the signal enhancer…”

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification

“…40,41 In combination with nanomaterials, such as gold nanoparticles (AuNPs), 27,42 graphene, 32,43 magnetic microparticles, 17,35,44,45 and quantum dots, 33 ultralow detection limits could be achieved with DSN-assisted signal amplification.…”